The invention relates to organogenesis, i.e. repair and/or neo-regeneration of tissues and organs in a mammalian body utilising autogenous stem cells of developed tissues. Particularly, the method of the invention is useful to regenerate or repair organs of endodermal origin such as bile duct, urinary bladder etc in vivo.
Some of the terms used in the specification are defined herebelow for better understanding and clarity.
1. xe2x80x98Organogenesisxe2x80x99: is a term Generally used in embryology; it denotes the formation of organs from the fertilised ovum in developing embryo.
2. xe2x80x98Neo-Regenerationxe2x80x99 in biological terms means restoration of lost structures of a tissue organ or the tissue/organ itself. As used herein, the term includes restoration of not only the tissue or structures thereof, but also an attempt to restore functions performed by the tissue/organ.
3. Histogenesis: Formation of tissues of the body.
4. Plasia: Growth or a change (Latinxe2x80x94To mould).
5. Metaplasia: Change of cells from normal to abnormal state (Latin-Meta+plasia). Thus, transformation of a fully developed tissue into another fully developed tissue is known as metaplasia.
6. Desired Metaplasia: It is a kind of Metaplasia, but the transformed tissue containing stem cells is useful, needed, desired and compatible anatomically, physiologically and histologically, in the new region of exposure and is responsible for neo-regeneration of tissues and organs.
7. Stem cells: These cells are a kind of primitive cells present in an embryo or an adult body and have the capacity to differentiate into specialised tissues, having different functions. These cells can be defined in terms of their functional capabilities. Stem cell is not a property but a spectrum of capability.
8. Regeneration: The growth of destroyed or devitalised tissue or organ from the remnant tissue or organ. It is a reparative attempt of the body. This regeneration is different from neo-generation as it does not involve formation total tissue or organ and is not by colonisation or grafting tissue or cells which in turn undergo transformation and form new tissue.
9. Neo-organogenesis: Formation of tissue or organ by the transformation of stem cells on colonisation by differentiation and proliferation.
10. Neo-histogenesis: Formation of tissues by differentiation and proliferation of stem cells on colonisation in the tissues.
11. Contiguous embryonal segment: It is the region in an embryo from where the donor and recipient tissue/organ have developed and the same region corresponds in the adult/developed body.
12. Adult or developed body: Animal body where embryonal morphogenesis is complete and is capable of independent existence even though same tissues/organs may be still developing or adulthood has reached. Hence, the term xe2x80x9cdeveloped bodyxe2x80x9d as used herein denotes a body in which embryonal morphogenesis is complete.
In nature, plant life and lower animals display the capacity to re-grow their lost tissues and organs. For example, newts can grow their amputated eye lens, lobsters and crabs can grow their broken claws, lizards can grow their severed tails and frogs can grow their amputated legs.
Basic laws of nature remain hidden in the revealed world of nature. It is known that a fertilised egg undergoes various stages of morphogenesis and develops into an embryo and eventually into an adult. Morphologically, the early embryos of different species are strikingly similar. At this early stage of development of embryo it is difficult to differentiate between the embryos of different species, and one cannot say which embryo will become fish, bird, dog, pig, monkey or man. Thus, a single celled ovum forms an adult body having different organs and tissues with different structure and function. Multicellular bodies of higher life forms are formed by differentiation of and proliferation of embryonal primitive germ cells. In order to sustain complex life functions, the cell specialisation has to be maintained and thus, organ regeneration capacity is compromised in higher forms of life due to specialisation of stem cells and maintenance of specialisation.
The laws of nature are difficult to understand but have uniform applicability, in nature. Therefore, if lower life forms can re-grow lost parts, the applicant believes that such capacity may be present in higher life forms as well. As such, there are numerous attempts in the prior art to regenerate tissues and organs, each with variable rates of success. Some attempts comprise isolation of tissues from pigs and culturing the same to regenerate organs like liver or kidneys, in vitro. The major disadvantage with in vitro procedures is that they are very expensive, and therefore, not always affordable. Besides, there is always a problem of revascularization and acceptance of the organ regenerated in vitro by the human body. In other words, organ transplantation though a successful process, is plagued by rejection phenomenon which needs life long use of immuno-suppressants. This not only increases the cost but jeopardises the host immunity. Non-availability of suitable donor, preservation and transportation of organ are a few other problems associated with organ transplantation. Hence, it is always preferable to exploit the body""s own potential to restore lost tissue/organs.
It is known that a fertilised ovum undergoes a series of divisions and forms a germ-layered disc. This disc consists of ectoderm, endoderm and mesoderm. These germ layers independently or in combination form various tissue and organs of an adult body. The endoderm of the embryonic germ disc forms a cavity lined by the cells of endodermal origin called xe2x80x98primary yolk sacxe2x80x99. A part of the cavity of the yolk sac is enclosed within the embryo to form the primitive gut, this gut is in free communication with the rest of the yolk sac. The part of the gut cranial (proximal) to this gut is the fore-gut; the part caudal is the hind-gut, while the intervening part is the mid-gut. The formation of these layers of the germ disc is attributed to the totipotent nature of the cells of the fertilized ovum. With the formation of the germ layers of germ disc, the cells lose their totipotent nature and become pluripotent, and as a consequence, these cells of the germ layers cannot form the whole body, but can only form certain tissues and body systems, derived from that specific germ layer. Thus, differentiation and specialisation of germ layer cells leads to specialised organ and tissues formation. Reverse activity i.e. formation of germ layer cells by the specialised tissue cells is not observed. Therefore, it is understood that the neo-regeneration of tissues requires pluripotent stem cells.
It is interesting to note that the stem cells developed from a particular layer of germ disc of developing embryo are present in some places in the developed tissues of adult body. These cells do maintain their pluripotent nature. In fact, xe2x80x9cstem cellsxe2x80x9d by their great proliferative property maintain differentiated cell populations throughout the life of an animal (Ashman L. 1999, Stem Cells Encyclopedia of Molecular Biol. Vol.4. Ed. By TE Geighton, Publishers: John Wiley and Sons Inc. NY, Chichester, Weinheim, Brisbane, Singapore, Torento P. 2430-2433). The large division potential makes these cells efficient tissue regenerators through clonal growth (C.S. Potten MS Loeffler. Stem Cells; Attributes, cycles, spirals, pitfalls and uncertainties, lessons, far and from the crypt Dev. 110-1001-1020=1990. Review article). However, the exact loci where these stem cells are situated in an adult or developed body is not known.
The Applicant worked and used the principle that stem cells still exist in the adult body and retain their capacity to form various tissues given the right kind of environment and stimuli. The applicant has noted that the stem cells present in the developed body have lost their capacity to migrate because the development in the adult body is fixed on localised. Hence, the applicant has used the inherent capacity of stem cells to proliferate, differentiate and form specialised tissues/organs in the required sites by transporting the stem cells to the desired sites by way of surgery. For this purpose, the applicant has first identified the embryonal germ layer in the embryo, from which the organ to be repaired or regenerated has developed. In other words, the applicant has identified the embryonal germ layer responsible for the origin of the organ desired to be regenerated or repaired. Then, the Applicant has identified the region in the developed body where the stem cells of that particular germ layer are situated. A membrane containing these cells is grafted onto the site or the organ which is to be regenerated or repaired. Using this principle, the Applicant has successfully regenerated various organs and tissues, developed from embryonic mesoderm in his earlier accepted and pending U.S. application Ser. No.08/921,307. The applicant has now used similar principles to regenerate various organs that have resulted from the differentiation and proliferation of pluripotent cells of the endoderm.
The main object of the invention is to provide an in vivo and in situ method for regeneration or repair of organs and tissues in mammals such as humans.
Another object of the invention is to provide an in vivo and in situ method for regeneration or repair of endodermal organs and tissues in mammals such as humans.
Another object of the invention is to provide a method for regeneration or repair of organs, eliminating the problems of organ transplantation such as non-availability of organs, rejection of transplanted organs by the recipient host, life long use of immunosuppressents by the recipient, need of perfect tissue matching etc.
Still another object is to provide a method for regeneration of organs, that utilises autogenous tissues.
One more object of the invention relates to effective management of diseases of organs.
Yet another object of the invention is to provide a cost effective method of regeneration of organs without any need for donor or costly equipments or costly nutrients to promote growth of cells.
Still another object of the invention relates to use of autogenous tissues so that the use of immunosuppressents is eliminated to prevent rejection phenomenon common in organ transplant surgery.
A further object of the invention is to utilise the phenomenon of xe2x80x98desired metaplasiaxe2x80x99 of tissues i.e. providing useful transformation of one tissue into required tissue(s).
Yet another object of the invention is for providing regeneration or repair of organ tissues employing stem cells in vivo.
One more object of the invention relates to the regeneration or repair of various organs/tissues of the body employing relevant stem cells from autogenous tissues present in various parts of the body.
Still another object of the invention relates to regeneration of organs/tissues by surgically transferring stem cells to the region of the organ/tissue system were regeneration is required.
Furthermore, the invention relates to regeneration of bile duct, urinary bladder from the embryonic contiguous regions of endoderm.
This invention relates to a method of isolating and developing a membrane containing pluripotent autogenous stem cells of endodermal origin for the use of neo-organogenesis and neo-histogenesis of various organs and tissues in mammals.
The invention relates to a method for repair or regeneration of organs and tissues that have originated from the endodermal plate of the germ disc of developing embryo. Thus, the invention provides an in-vivo method of neo-organogenesis of various tissues or organs developed from endodermal germ layer, in a mammalian body, comprising the steps of surgically transferring an autogenous membrane containing stem cells to the site of the organ or tissue of endodermal origin, to be regenerated, said membrane being selected from the corresponding contiguous embryonal segment in a developed body and providing the functional need and tissue environment for regeneration of the desired tissue or organ.
In general, the method in vivo and in situ for regeneration or repair of organs, comprises the following steps:
i) identifying the embryonal germ layer responsible for the origin of the organ to be regenerated or repaired, and the corresponding contiguous embryonal segment in a developed body,
ii) isolating an autogenous membrane containing pluripotent stem cells from the identified site, wherein the stem cells in the membrane are rendered free of the influence of the local anatomical tissue environment,
iii) surgically transferring the said autogenous membrane to the region where repair or neo-regeneration of the organ is desired, and
iv) providing appropriate stimuli and sufficient time for differentiation and proliferation for neo-regeneration.
The tissue from which the autogenous cellular membrane is obtained may be termed as the xe2x80x98donor tissuexe2x80x99 for ease of reference and the tissue or organ to be regenerated or to which region or locus, the donor tissue cells are grafted or colonised, may be termed as xe2x80x98recipient tissuexe2x80x99.
An important aspect is that the donor and recipient tissues should essentially be derived from the same germ layer in an embryo. Each of the tissues and organs of the human body owe their origin to different germ layers of the germ disc of the embryo. For example protective tissues viz. Skin and nerve tissues are ectodermal derivatives, lining of the epithelium and exocrine glands originate from the endoderm, while cardiovascular, genito-urinary and other systems are formed from the mesoderm. Similarly, the gut originates from the endoderm. Parts of the gut such as the trachea also originate from the endoderm. It is observed that the donor and recipient tissues must be selected from the neighbouring regions in a developing embryo. This enables proper neo-regeneration. Graft survival is possible only when donor and recipient tissues in question originate from the same geim layer in the embryo. Stem cells"" response to Extra Cellular Matrix cytokine signalling is matched and compatible. Therefore, for desired metaplasia to take place, cell potential, environment and functional need are all essential. In the present invention, the organs to be repaired or regenerated develop from the endodermal layer of the germ disc of the embryo.
The donor tissue selected is such that it is replete with stem cells preferably of pluripotent nature. If the cells do not have the potential to differentiate and proliferate in different path ways, desired metaplasia may not initiate at all and neo-regeneration of total tissue or organ may not be possible. The autogenous membrane or the donor tissue is characterised by the presence of stem cells which when exposed to the environment of the recipient tissue, are capable of reacting with tissue organisers and enable regeneration of organs. The autogenous membrane isolated from the identified site is characterised in that it is rendered free from influence of local anatomical tissue environment and are made available for differentiation and proliferation in the site where regeneration/repair of the organ is desired. Thus, the stem cells become readily receptive to the new environment which has been provided by surgical transfer i.e. the site of exposure (site of recipient tissue) where repair or neo-regeneration is desired. The stem cells of the autogenous membranes are made susceptible to the action of the local tissue organisers (induces, inhibitors, etc.). Pluripotent nature of stem cells helps in formation of all the histological components of the tissue or organ. Stem cells have capacity to survive indefinitely and independently in the tissues. These cells have coded memory in RNA and can synthesize their own new protein and thus are capable of differentiation and proliferation in different pathways. The intrinsic factors such as messenger gene and genetic factors, inherent in the stem cells is exploited for regeneration or repair of the desired organ. Accordingly, these cells have potential for neo-regeneration of tissues. These cells during differentiation also maintain stem cell character in one progeny while other is differentiating. Therefore, presence of stem cells in donor tissue is an important criteria for neo-regeneration.
When the autogenous cellular membrane is transferred to the site of repair/neo-regeneration of the concerned organ, the said membrane is faced with a new environment with new functional needs. The step of providing functional need includes creating a stress of new functional need of the tissue system of new location to which stem cells have been shifted, to induce desired metaplasia. It is the applicants"" finding that the cytokines in the region of the recipient tissue acton the cells of the autogenous membrane, especially the stem cells, and thus trigger the differentiation and proliferation of the stem cells in the membrane into desired cells required in the region, resulting in the neo-regeneration or repair of the organ. Some of the factors responsible for stem cell proliferation are messenger genes, genetic factors, instrinstic capacity and messages coded in the stem cells.
Cell surface receptors have specificity of response to specific cytokines. Therefore, compatibility of Cell Surface Receptors of the donor tissue should match with that of the surrounding tissues (recipient tissues).
Time: Time is yet another important factor. Once the donor tissue is grafted to the recipient time or organ, the graft should be allowed to develop/mature for a period sufficient to enable regeneration of organs. The period varies from organ to organ: in general, a period of at least 3 to 5 months should be allowed for the organ to regenerate/develop. This is because time is taken for cellular response to exhibit the outcome. This is needed for synthesis of required protein, differentiation and proliferation, etc.
The organ regenerated contains all the layers of cells as in the original organ. The shape and histological structure of the regenerated organ is also found to be identical to that of the lost original organ. The organ so regenerated or repaired is capable of performing all its functions.
In an embodiment the muscosal surface of isolated membrane is kept towards the lumen of the organ to be regenerated/repaired. Also, a support is placed inside the autogenous membrane to counter act the abdominal pressure exerted. The ends of the tubular graft and the CBD are spatulated before anastomosis to avoid constriction at the anastomosed site.
In another embodiment the organogenesis of various tissues/organs incorporates functions of tissue inducers and/or tissue organisers to achieve regeneration/repair of any tissue or organ into its proper size, shape and form to perform its inherent function.
The Applicant has successfully used the aforementioned principles to regenerate several organs that owe their origin to the embryonic endoderm. Two specific examples are illustrated hereinxe2x80x94the regeneration of bile duct and urinary bladder. These examples should not be construed as limitations on the inventive scope embodied herein.
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